Novel phenylboronic acid modified chitosan microcarrier, preparation method and application

Abstract

The invention relates to the technical field of biology, in particular to a novel phenylboronic acid modified chitosan microcarrier, a preparation method and application, and the phenylboronic acid modified chitosan microcarrier is obtained by mixing 4-formyl phenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking. The particle size of the microcarrier is about 100 [mu] m, and scanning electron microscope analysis shows that the microcarrier shows a clear spherical porous form, has uniform pore shape and uniform pore size, and can provide a three-dimensional environment for cell growth. The prepared porous material has moderate hardness, elasticity and pore wall, is more beneficial to growth of more cells and transmission of nutrient substances, has higher stability, is more suitable for cell culture, shows excellent biological safety and biocompatibility, and is more suitable for a 3D cell culture system.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a novel phenylboronic acid-modified chitosan microcarrier, its preparation method, and its application. Background Technology

[0002] The creation of spheroids and organoids derived from human stem cells is a crucial step in addressing numerous medical, pharmacological, and biological challenges. Traditional culture methods are performed on two-dimensional planar culture dishes, where cells adhere to artificial plastic or glass substrates, contacting other cells only at their periphery. Due to the lack of oxygen, nutrient, or waste gradients, the environment is physiologically non-uniform. Cells are not allowed to stack together but are forced into a monolayer morphology, which is not the natural morphology for all cell types. Establishing co-cultures in petri dishes can increase natural contact and communication between cells, but the two-dimensional surface still inhibits the ability of cells to form multidimensional structures. Therefore, 2D cultured planar cell layers cannot accurately mimic the growth environment of cells in vivo. With technological advancements, 3D suspension cell culture has become a key step in constructing organoids. 3D suspension cell culture, based on biological scaffold materials, allows for the formation of three-dimensional organoid structures through cell-driven self-assembly or microenvironment regulation, better mimicking the morphology and physiology of cells.

[0003] Microcarriers, as biological scaffolds for suspended cell culture, provide a stable matrix microenvironment for cells due to their porous structure and inherent material properties, playing a crucial role in cell proliferation, drug delivery, and organoid construction. Compared to two-dimensional cell culture, three-dimensional cell culture based on microcarriers can simulate the in vivo microenvironment, enabling rapid cell proliferation, enhancing stem cell stemness and directed differentiation capabilities, and facilitating temporal cell regeneration, thereby guiding temporal functional changes in stem cells. Currently available cell microcarriers can achieve large-scale cell expansion to a certain extent, but various enzymes are needed to degrade the microcarriers to obtain single cells. Furthermore, in in vivo delivery scenarios, the degradation of microcarriers and the sustained release of cells face significant bottlenecks. Therefore, developing a novel microcarrier capable of spontaneously degrading in response to tissues, organs, and the body environment has profound social and medical significance for stem cell expansion, improved stem cell performance, and stem cell delivery.

[0004] To address the aforementioned issues, this invention develops a chitosan (CS-FPBA) microcarrier synthesized using an emulsification method and modified with phenylboronic acid. This microcarrier not only enables large-scale cell expansion but also possesses glucose and pH-sensitive properties. It can self-degrade in vivo and in individuals with specific diseases (diabetes) to achieve the delivery and sustained release of stem cells. Summary of the Invention

[0005] The primary objective of this invention is to provide a novel chitosan microcarrier modified with phenylboronic acid, wherein the chitosan microcarrier is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking them.

[0006] Preferably, the particle size of the phenylboronic acid-modified chitosan microcarrier is >100 μm.

[0007] A second objective of this invention is to provide a method for preparing the phenylboronic acid-modified chitosan microcarrier, comprising the following steps: (1) Medium-viscosity chitosan is dispersed in acetic acid solution, fully dissolved, and activated; (2) Slowly add 4-formylphenylboronic acid to the solution obtained in step (1) and stir at room temperature; (3) Dialyze, adjust pH to 5.0 to remove excess 4-formylphenylboronic acid, freeze dry to obtain product CS-FPBA; (4) The product CS-FPBA obtained in step (3) is fully dissolved, and gelatin and crosslinking agent are added by stirring to form a reaction system; (5) Stir the liquid paraffin and Span 80 until they are evenly mixed to form a solvent system; (6) The reaction system described in step (4) is slowly added to the solvent system described in step (5) under stirring, the reaction is carried out, the mixture is allowed to stand, and the precipitate is collected. (7) Cleaning step (6) The precipitate collected is swollen with ultrapure water, then frozen and dried to obtain the final product CS-FPBA microcarrier.

[0008] Preferably, the concentration of 4-formylphenylboronic acid in step (2) is 0.02 mmol.

[0009] Preferably, the crosslinking agent in step (4) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0010] Preferably, the gelatin concentration in step (4) is 5%.

[0011] Preferably, the volume-to-mass ratio of the liquid paraffin and Span 80 in step (5) is 250:5.

[0012] Preferably, the reaction temperature in step (6) is 30°C and the reaction time is 48h.

[0013] Preferably, the cleaning in step (7) uses isopropanol and anhydrous ethanol.

[0014] A third objective of this invention is to provide the application of the phenylboronic acid-modified chitosan microcarriers or the phenylboronic acid-modified chitosan microcarriers prepared by the aforementioned preparation method in three-dimensional cell culture.

[0015] The beneficial effects of the present invention are: (1) The present invention provides a novel chitosan microcarrier modified with phenylboronic acid, wherein the chitosan microcarrier modified with phenylboronic acid is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking. The particle size of the microcarrier is about 100 μm. Scanning electron microscopy analysis shows that the microcarrier exhibits a clear spherical porous morphology with uniform pore shape and uniform pore size, which can provide a three-dimensional environment for cell growth. Its hardness, elasticity and pore wall are moderate, which is more conducive to the growth of more cells and the transport of nutrients.

[0016] (2) At pH 5.5, the degradation rates of both CS and CS-FPBA were relatively high; at pH 6.5, the degradation rate of CS was lower than that of CS-FPBA; at pH 7.4, the degradation rate of CS-FPBA was lower than that of CS. In summary, CS-FPBA has higher stability. The optimal pH range for animal cell culture is between 7 and 7.5, and CS-FPBA is more suitable for cell culture applications. Under the same culture mode, cell proliferation in the CS-FPBA microcarrier environment was significantly better than that in CS.

[0017] (3) Optical microscopy confirmed that cells adhered well to the porous structure of the microcarrier. Cells on the microcarrier showed significant proliferation and formed tight junctions within 1-9 days, and the expression rate of SHED in 3D cultured SHED (SHED-CD) was significantly increased compared with that of 2D cultured SHED. After culturing SHED on CS-FPBA microcarriers and co-culturing them in a stirred bioreactor, assembled SHED microcarriers were obtained, and SHED maintained good biological activity and differentiation potential. Therefore, CS-FPBA microcarriers exhibit excellent biosafety and biocompatibility, and are more suitable for 3D cell culture systems. Attached Figure Description

[0018] Figure 1 Schematic diagram of the synthesis process of phenylboronic acid-modified chitosan CS-FPBA microcarriers Figure 2 CS-FPBA macroscopic image and microscopic structure image Figure 3 In vitro degradation rate of CS and CS-FPBA Figure 4 Stability of CS and CS-FPBA at different pH values Figure 5 The states of CS and CS-FPBA in different solutions Figure 6 CS and CS-FPBA cell proliferation assay Figure 7 Application of CS-FPBA microcarriers for cell culture Figure 8 CS-FPBA cell culture scanning electron microscope Figure 9 CS-FPBA cell proliferation assay Figure 10 SHED cells cultured on CS-FPBA microcarriers still maintain the stem cell characteristics of mesenchymal stem cells. Figure 11 SHED cells cultured on CS-FPBA microcarriers exhibit excellent osteogenic capacity. Detailed Implementation The scope of protection of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the scope of protection of the present invention is not limited by the following embodiments.

[0019] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0020] Example 1: A method for preparing a chitosan carrier Includes the following steps: (1) Medium viscosity chitosan (CS) was dispersed in 1% acetic acid solution and fully dissolved to obtain a stable chitosan alternating suspension; (2) Freeze-dry to obtain product CS; (3) Place CS in an oven to dry for 40 min, soak it in 70% ethanol for 24 h, and wash it repeatedly with deionized water 3 times. (4) After fully dissolving the 1% synthetic product CS obtained in step (3), add 0.5% gelatin while stirring, and add crosslinking agent (EDC+NHS) to form a reaction system; (5) 250 mL of liquid paraffin + 5 g Span 80, stir at 330 rpm until the mixture is homogeneous, which is the solvent system; (6) The reaction system described in step (4) is slowly added to the solvent system described in step (5) under stirring; (7) After reacting at 30℃ for 48 h, let stand and collect the precipitate; (8) Wash with isopropanol and anhydrous ethanol in sequence, swell with ultrapure water, freeze and dry to obtain the final product CS microcarrier, and store it in a dry place.

[0021] Example 2: A method for preparing a phenylboronic acid-chitosan carrier Includes the following steps: (1) Medium viscosity chitosan (CS) was dispersed in acetic acid solution, fully dissolved, and activated for 4 hours; (2) Slowly add 0.02 mmol of 4-formylphenylboronic acid (FPBA) to the mixed solution described in step (1) and stir at room temperature for 12 hours; (3) Dialyze the dialysis bag with distilled water for 3 days, adjust the pH to 5.0, and remove excess FPBA; freeze dry to obtain the product CS-FPBA for later use; (4) After the 1% synthetic product CS-FPBA obtained in step (4) is fully dissolved, 0.5% gelatin is added under stirring, and crosslinking agent (EDC+NHS) is added to form the reaction system; (5) 250 mL of liquid paraffin + 5 g Span 80, stir at 330 rpm until the mixture is homogeneous, which is the solvent system; (6) The reaction system described in step (4) is slowly added to the solvent system described in step (5) under stirring; (7) After reacting at 30℃ for 48 h, let stand and collect the precipitate; (8) Wash with isopropanol and anhydrous ethanol in sequence, swell with ultrapure water, freeze and dry to obtain the final product CS-FPBA microcarrier, screen microcarriers with a particle size >100 μm and dry and store. Chitosan is a rapidly developing medical polymer material with characteristics such as low immunogenicity, controllable degradation, and porosity. To manufacture a multifunctional chitosan-based scaffold that supports stem cell proliferation and differentiation, we added 4-formylphenylboronic acid to obtain a microcarrier material that can be used for cell culture and enhance cell differentiation.

[0022] Preparation process as follows Figure 1 As shown, after dialysis, CS-FPBA was obtained as a dry powder through freeze-drying. Figure 2 A), and stored in a desiccator under ambient conditions for subsequent application. It can be seen that CS-FPBA microcarriers with a particle size of approximately 100 μm were prepared. Figure 2 (B) Scanning electron microscopy analysis showed that the microcarriers exhibited a distinct spherical porous morphology with uniform pore shape and size, providing a three-dimensional environment for cell growth. Their moderate hardness, elasticity, and pore wall structure further facilitated cell growth and nutrient transport.

[0023] Example 3: Determination of the in vitro degradation rate of microcarriers The degradation performance of microcarriers was studied using PBS buffer at room temperature. First, the dried carrier material was weighed (mass denoted as m1). Then, the sample material was immersed in PBS buffer and incubated at 37°C for 40 days. At specified time intervals, the sample was removed and washed with deionized water. Finally, the rinsed sample was lyophilized a second time and weighed again (mass denoted as m2).

[0024] The degradation rate of the stent is calculated using the following formula: DR = (m1 m2) / m1×100% The in vitro degradation rates of CS and CS-FPBA scaffolds after immersion in PBS for 40 days were as follows: Figure 3 As shown, the in vitro degradation rate of CS-FPBA was lower than that of CS, indicating that the chitosan-phenylboronic acid microcarriers prepared in this experiment degraded slowly, suggesting that the chitosan-phenylboronic acid microcarriers can maintain cell adhesion, spreading, and growth metabolism on the surface of the microcarriers.

[0025] High-quality microcarriers require adaptability to various culture systems and a certain degree of stability. According to... Figure 4 It can be seen that at pH 5.5, the degradation rates of both CS and CS-FPBA materials are relatively high; at pH 6.5, the degradation rate of CS is lower than that of CS-FPBA; and at pH 7.4, the degradation rate of CS-FPBA is lower than that of CS. In summary, CS-FPBA has higher stability. The materials were immersed in solutions at different pH values, and their integrity was observed. Figure 5 The optimal pH range for animal cell culture is between 7 and 7.5. Based on observations, CS-FPBA is more suitable for cell culture applications.

[0026] Example 4: Cell Proliferation Experiment After obtaining two microcarrier materials, they were applied to cell culture and the cell proliferation was observed. A three-dimensional culture mode was constructed using a bioreactor, with 100 mg of microcarriers at 2.5 × 10⁻⁶ cells / year. 6 Cell culture was seeded at the following rates: On the first day, 50 mL of culture medium was inoculated and cultured at a variable speed (40 rpm) for 24 hours. On the second day, the culture medium was increased to 75 mL and cultured at a constant speed. Cells were then stained and observed using a fluorescence microscope, avoiding light during the staining process. Figure 6 It can be seen that, under the same culture mode, cell proliferation in the CS-FPBA microcarrier environment is significantly better than that in CS.

[0027] Example 5 2D cell culture methods: In a clean cell culture bench, place the tissue in a culture dish and rinse repeatedly with PBS until no obvious blood is visible on the surface. Discard the PBS, add 1.5 ml of collagenase to the culture dish, cut the tissue into pieces, and digest in a 5% CO2 cell incubator. Add an equal volume of culture medium and centrifuge at 800 rpm for 5 min. Seed the tissue to a height of 25 cm. 2 After mixing the culture flasks, place them in a cell incubator for culture; discard the original culture medium, wash three times with sterile PBS; add trypsin for digestion for 3 min; add an appropriate amount of culture medium, pipette and transfer to a 15 ml centrifuge tube for centrifugation; discard the supernatant, resuspend the dental pulp stem cells (SHED) and then add 1×10⁻⁶ cells / mL. 6 / bottle inoculated at a density of 75 cm 2 Place in a culture flask and set aside for later use.

[0028] 3D cell culture methods: The synthesized microcarriers were sterilized under ultraviolet light in a sterile biosafety cabinet for 6 hours. The sterilized microcarriers were then transferred to 125 mL breathable culture flasks with built-in impellers, and culture medium was added. The culture flasks were then placed on a 3D mini bioreactor and rotated at a uniform speed to ensure complete swelling of the microcarriers. Figure 7 Dental pulp stem cells (SHED) were used at a rate of 2.5 × 10⁻⁶. 6 Cells were seeded at a concentration of [cell density] / mL into culture flasks containing microcarriers, and culture medium was added to a final volume of 50 mL, with a 24-hour incubation period. The next day, the culture medium was increased to 75 mL, and the cells were cultured in a constant rotation mode. SHED-microcarrier assemblies were then obtained for subsequent experiments. 3D cultured SHEDs were obtained by digestion with 0.25% trypsin at 37°C for 5 min.

[0029] Protein sample preparation and detection methods: Protein samples from 2D and 3D cultured SHED were prepared by treating with RIPA lysis buffer at 4 °C for 20 min. The samples were centrifuged for 15 min, and the supernatant was collected for protein concentration quantification using a BCA protein assay kit. Equal volumes of protein samples were mixed with loading buffer and PBS and loaded onto a protein electrophoresis precast gel for protein validation.

[0030] Optical microscopy confirmed that cells adhered well to the porous structure of the microcarrier. Scanning electron microscopy was used to observe the morphology and structure of the 3D cultured cell spheroids at different time points. Observations showed that cells on the microcarrier exhibited significant proliferation and formed tight junctions within 1-9 days. Figure 8 By using cells cultured in a two-dimensional manner as a control group, a significant increase in cell count was observed throughout the culture period. Figure 9A). Staining results showed that the expression rate in 3D cultured SHED (SHED-CD) was significantly higher than that in two-dimensional cultured SHED. Figure 9 B). SHED was cultured on CS-FPBA microcarriers and co-cultured in a stirred bioreactor to obtain assembled SHED microcarriers. The SHEDs maintained good biological activity and differentiation potential. Figure 10 Our study further confirms that after establishing a three-dimensional culture system using microcarriers and SHED suspension culture, SHED highly expresses osteogenic development-related proteins such as RUNX2. Figure 11 This allows it to better maintain its developmental potential and exhibits excellent osteogenic capacity. Therefore, these results indicate that the CS-FPBA microcarrier demonstrates excellent biosafety and biocompatibility, making it more suitable for 3D cell culture systems.

[0031] In summary, this invention provides a novel chitosan microcarrier modified with phenylboronic acid. The phenylboronic acid-modified chitosan microcarrier is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and cross-linking the mixture. The microcarrier has a particle size of approximately 100 μm. Scanning electron microscopy analysis shows that the microcarrier exhibits a distinct spherical porous morphology with uniform pore shape and size, providing a three-dimensional environment for cell growth. Its moderate hardness, elasticity, and pore wall properties are more conducive to cell growth and nutrient transport. At pH 5.5, both CS and CS-FPBA showed high degradation rates; at pH 6.5, the degradation rate of CS was lower than that of CS-FPBA; and at pH 7.4, the degradation rate of CS-FPBA was lower than that of CS. Therefore, CS-FPBA exhibits higher stability. The optimal pH range for animal cell culture is between 7 and 7.5, making CS-FPBA more suitable for cell culture applications. Under the same culture conditions, cell proliferation under the CS-FPBA microcarrier environment was significantly better than that under CS. Optical microscopy confirmed good cell adhesion on the porous structure of the microcarriers. Cells on the microcarriers exhibited significant proliferation and formed tight junctions within 1–9 days, with a significantly higher expression rate in 3D-cultured SHED (SHED-CD) compared to 2D-cultured SHED. After culturing SHED on CS-FPBA microcarriers in a stirred bioreactor, assembled SHED microcarriers were obtained, and SHED maintained good bioactivity and differentiation potential. Therefore, CS-FPBA microcarriers exhibit excellent biosafety and biocompatibility, making them more suitable for 3D cell culture systems.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A novel phenylboronic acid-modified chitosan microcarrier, characterized in that, The phenylboronic acid modified chitosan microcarrier is obtained by mixing and cross-linking 4-formylphenylboronic acid and medium viscosity chitosan with gelatin.

2. The phenylboronic acid-modified chitosan microcarriers of claim 1, wherein, The particle size of the phenylboronic acid modified chitosan microcarrier is greater than 100 μm.

3. The method for preparing chitosan microcarriers modified with phenylboronic acid as described in claim 1, characterized in that, The method comprises the following steps: (1) dispersing medium viscosity chitosan in acetic acid solution, fully dissolving and activating; (2) slowly adding 4-formylphenylboronic acid into the solution obtained in step (1) and stirring at room temperature; (3) dialyzing, adjusting pH to 5.0, removing excess 4-formylphenylboronic acid, freeze-drying to obtain the product CS-FPBA; (4) fully dissolving the product CS-FPBA obtained in step (3), stirring to add gelatin and cross-linking agent to form a reaction system; (5) stirring liquid paraffin and Span 80 until they are uniformly mixed to form a solvent system; (6) slowly adding the reaction system in step (4) into the solvent system in step (5) under stirring, reacting, standing and collecting the precipitate; (7) washing the precipitate collected in step (6), swelling in ultrapure water, freezing and drying to obtain the final product CS-FPBA microcarrier.

4. The production method according to claim 3, wherein The concentration of 4-formylphenylboronic acid in step (2) is 0.02 mmol.

5. The production method according to claim 3, wherein The cross-linking agent in step (4) is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

6. The production method according to claim 3, wherein The concentration of gelatin in step (4) is 5%.

7. The production method according to claim 3, wherein The volume / mass ratio of liquid paraffin and Span 80 in step (5) is 250:

5.

8. The production method according to claim 3, wherein The reaction temperature in step (6) is 30℃ and the reaction time is 48h.

9. The production method according to claim 3, wherein The washing agent in step (7) is isopropanol and anhydrous ethanol.

10. The phenylboronic acid modified chitosan microcarrier of claim 1 or prepared by the method of any one of claims 3-9 for use in three-dimensional cell culture.

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